Nanotechnology

1. Nanotechnology • Development and use of materials and devices for analysis and measurement on a nanometer scale • Diverse and interdisciplinary field : Biology/Biotechnology,Physics, Chemistry, Material sciences, Electronics, Chemical Engineering, Information technology • Future implications - Creation of new materials and devices in medicine, electronics, energy etc. - Social issues: toxicity, environmental impact of nano-materials

2. Nanotechnology Plays by Different Rules Normal scale Nanoscale

3. Nano-sized materials • Unusual and different property - Semiconductor nanocrystals : Size-dependent optical property: Absorption and emission - Magnetic nanoparticles (iron oxide) Ferromagnetic: Materials that can be magnetized by an external magnetic filed and remain magnetized after the external field is removed The spin of the electrons in atoms is the main source of ferromagnetism Super-paramagnetic : In the absence of magnetic field  Magnetization is zero An external magnetic field is able to magnetize the nanoparticles ex) MRI contrast agents

4. Nano-Bio Convergence Bio-inspired device and system Bio-Technology Nano-Technol Molecular Imaging Molecular Switch DNA barcode Biochip / Biosensor Nanotherapy / Delivery Bionano-machine / Nano-Robot

5. Nano-Biotechnology • Integration of nano-sized/structured materials, nano-scale analytical tools, and nano-devices into biological sciences including biotechnolgy for development of new biomaterials and analytical toolkits • Use of bio-inspired molecules or materials - Typical characteristics of Biological events - Self assembly - Highly efficient : high energy yield - Very specific : extremely precise • Bio-molecules DNA, Proteins, Antibody, RNA, Aptamers,, Peptides,

6. Applications and perspectives of nanobiotechnology • Development of tools and methods -More sensitive - More specific - Multiplexed - More efficient and economic • Implementation -Diagnosis and treatment of diseases : Nanomedicine Rapid and sensitive detection(Disease biomarkers, Imaging), Targeted delivery of therapeutics - Drug discovery - Understanding of life science

7. Examples • Nano-Biodevices • Nano-Biosensors • Biomolecular motors • Medical use : Targeting with Ab-magnetic beads Contrast agents for MRI • Analysis of a single molecule/ a single cell

8. Issues to be considered • Synthesis or selection of nano-sized/ structured materials : Biocompatibility, cytotoxicity • Functionalization with biomolecules or for biocompatibility • Integration with devices and/or analytical tools • Assessment : Reproducibility, Toxicity • Implementation to human body

9. The size of Things

10. 이전까지 방식은 이미 존재하는 큰 물질을 깎아서 원하는 작은 크기로 만드는 것 : top-down 나노 물질은 원자나 분자를 벽돌처럼 쌓듯이 조합해 완전히 새로운 방식 : bottom-up Top-down : Hard to fabricate materials less than 50nm in size Bottom-up : Self-assembly 현재 생체분자처럼 원자나 분자가 알아서 어떤 물질로 만들어지는 방법인 자기조립이 활발히 연구 중. 사진은 원자들이 일정 형태를 갖추는 자기조립의 과정을 보여 줌 How are nano-sized materials fabricated ?

11. NanoBiotech was initiated by development of AFM and STM that enable imaging at atomic level in 1980

12. Atomic Force Microscope Foremost tool for imaging, measuring, and manipulating matter at the nanoscale - When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law - Deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes • - If the tip was scanned at a constant height, a risk would exist that the tip collides with the surface, causing damage. • A feedback mechanism is employed to adjust the tip-to-sample distance to maintain a constant force between the tip and the sample. • Sample is mounted on a piezoelectric tube that can move the sample in the z direction for maintaining a constant force - Contacting mode - Non-contacting mode - Tapping mode

14. Image of ATP synthase composed of 14 subunits Example showing the resolution of protein structure by AFM

15. Optical Properties Of Quantum Dots b) Photostability a) Multiple colors d) High quantum yield c) Wide absorption and narrow emission Quantum Yield ≥ 60 ~ 70 % Single source excitation

16. Y Y Y Y Y Y Y Y Y Y QD QD Y Y Y Y Organelle Y Y Organelle In Vivo Cell Imaging + QD-Antibody conjugates Antigen ▲ 3T3 cell nucleus stained with red QDs and microtubules with green QDs - Multiple Color Imaging - Stronger Signals Wu et al. Nature Biotech.2003 21 41

17. In Vivo Cell Imaging Live Cell Imaging Quantum Dot Injection ▶ Red Quantum Dot locating a tumor in a live mouse Cell Motility Imaging 10um ◀ Green QD filled vesicles move toward to nucleus (yellow arrow) in breast tumor cell Alivisatos et al., Adv. Mater., 2002 14 882

18. Förster (or Fluorescence)ResonanceEnergy Transfer (FRET) • Non-radiative energy transfer from an energy donor to an energy acceptor • Dipole –dipole coupling • Energy transfer efficiency : • - Degree of spectral overlap between donor fluorescence emission and acceptor • absorption • - ~ 10 nm

19. Use of FRET measurements Molecular ruler in the determination of inter- or intra-molecular distances • Detection of target analytes • Analysis of biomolecular interactions • Single molecule analysis • - Protein folding/unfolding • - Protein dynamics • - The transfer efficiency is dependent on the inter-fluorophore distance. • Calculation of the transfer efficiency allows the distance between fluorophores.

20. QDs as an energy donor - High quantum yield (~0.6) and less photo-bleaching Strong PL intensity - Multiple acceptors per single QD Increased ET efficiency - Narrow and size-tunable emission Multiplexed assay AuNPs as an energy acceptor (Quencher) - Higher quenching efficiency than conventional quenchers - Extended working distance by SET mechanism : ~ 22 nm - Applicable to various donors FRET probe based on Quantum dots and AuNPs Oh et al., JACS (2006) Oh et al., Angewandte Chemi Intl Ed.(2007)

21. Förster Resonance Energy Transfer (FRET) using QDs Excitation Emission Excitation FRET quenching QD QD quencher QD quencher QD QD with Quencher PL intensity Time (ns)

22. Protease Assay • Protease • A variety of proteases in cells • Essential for the dynamic regulations of cell function and aberrations • Involved in major human diseases (cancers, apoptosis, and inflammation) • Protease inhibitors are known to be drug candidates • Matrix metalloproteinases (MMPs), Caspase-3, Thrombin • Conventional Methods for Protease Assay • Gel- and LC-MS-based methods: accurate, but need a long analysis time • Limitation to high throughput assay Simple, fast, and high throughput method is required “ Assay of proteases using FRET probe” - Simple and sensitive - Multiplexed and high-throughput assay

23. Matrix metalloproteinases (MMPs) • Regulate cancer metastasis by degrading ECM components or cytokines • MMP-7 : Potential biomarker ofhuman colorectal or breast cancer • MMP-2 & 9 : Human carcinoma • MMP-3 : Joint damage • MMP inhibitors : Candidates for anticancer therapeutics Steps in the process of metastasis Nat. Rev. Cancer 2002, 2, 161

24. Au Construction of FRET probe for Protease Assay Peptide-conjugated AuNP cysteine 1.4 nm in diameter biotin + Au monomaleimide AuNP peptide FRET probe based on QDs and AuNPs Kim et al., Anal. Chem. (2009)

25. PL  PL  PL  Wavelength  Wavelength  Wavelength  Chip-based assay dequenching + protease Glass Glass quenching quenching + protease + inhibitor Glass Kim et al., Anal. Chem. (2009)

26. Chip-based Protease Assay Silver-staining Fluorescence Relative PL (%) SA-QD605 (A) SA-QD605 +Pep-AuNPs (B) SA-QD605 +Pep-AuNPs +MMP-7 protease (C) SA-QD605 +Pep-AuNPs +MMP-7 protease +Inhibitor (D)

27. Specificity of FRET probe + Thrombin + Caspase-3 + MMP-7 Peptide sequences for proteases Thrombin : Biotin-GKGGLVPR-GSGC MMP-7 : Biotin-KSRWLA-LPRC Casp-3 : Biotin-GRRGDEVD-GGGRRC

28. α -Hemolysin: Self-Assembling Transmembrane Pore • A self-assembling bacterial exo-toxin produced by some pathogens like Staphylococcus aureus as a way to obtain nutrients  lyzes red blood cells • Alpha-hemolysin monomers bind to the outer membrane of susceptible cells. • The monomers oligomerize to form a water-filled heptameric transmembrane channel that facilitates uncontrolled permeation of water, ions, and small organic molecules. • Rapid discharge of vital molecules, such as ATP, dissipation of the membrane potential and ionic gradients, and irreversible osmotic swelling leading to the cell wall rupture (lysis), can cause death of the host cell.

29. - Mushroom-like shape with a cap and stem • 50 A bets-barrel stem • Narrowest part : ~ 1.4 nm in diameter

30. Biotechnological applications • Unique structural features make self-assembling membrane channel suitable for biotechnological applications • Assembled alpha-hemolysin : stable over a wide range of pH and temperature • Transmembrane pore stays open at normal conditions • Hemolysin binds to various biological or synthetic lipid bilayer • Delivery system : facilitate controlled delivery of ions and small organic compounds such as sugars and oligonucleotides across the plasma membrane of cells or through the walls of synthetic lipid vesicles : Engineered pores • Stochastic sensors • DNA sequencing

31. Stochastic sensor • A molecular adaptor is placed inside its engineered stem, • influencing the transmembrane ionic current induced by an applied voltage • Reversible binding of analytes to the molecular adaptor transiently • reduces the ionic current • Magnitude of the current reduction : type of analyte • Frequency of current reduction : analyte concentration

32. Stochastic Sensors

33. a: Histidine captured metal ions (Zn+2, Co+2, mixture ) b: CD captures anions ( promethazine, imipramine, mixture)c : biotin ligand

34. DNA sequencing - Transmembrane potential drives translocation of DNA or RNA through the pore - Ionic current blockades reflect the chemical structure of individual strands - Discrimination of different sequences of DNA or RNA - Single resolution of purine and pyrimidine nucleotides Eelectrophoretically-driven translocation of a 58-nucleotide DNA strand through the transmembrane pore of alpha-hemolysin

35. Single DNA Engineering 유전자의 일부가 잘못되었을 때 잘못된 유전자를 제거하고 정상적인 유전자로 대체. 단일 DNA를 고정하기 위해서 광학적 포획(optical trapping)기술을 이용 DNA의 양단에 구슬(bead)을 붙이고 레이저를 이용하여 구슬을 잡으면 수용액상에서 끊임없이 움직이는 단일 DNA를 고정시킬 수 있음. 자르고 싶은 위치에 마이크로피펫(micropipet)을 이용 제한효소를 넣어 DNA 조각을 잘라냄. 잘린 DNA는 전극을 이용하여 펴고 정상적인DNA를 넣어 효소를 이용하여 DNA를 연결함. 단일 DNA는 형광물질로 염색 시키고, CCD카메라가 장착된 형광현미경을 이용하여 관찰.

36. Field Effect Transistor A transistor is a linear semiconductor device that controls current with the application of a lower-power electrical signal. The bipolar and field-effect transistors. The bipolar transistors utilize a small current to control a large current. The field-effect transistor utilizes a small voltage to control current : The junction field-effect transistor.

37. In a junction field-effect transistor, or JFET, the controlled current passes from source to drain, or from drain to source as the case may be. The controlling voltage is applied between the gate and source. With no voltage applied between gate and source, the channel is a wide-open path for electrons to flow. However, if a voltage is applied between gate and source of such polarity that it reverse-biases the PN junction, the flow between source and drain connections becomes limited, or regulated.

39. Carbon Nanotube FET Chen et al., PNAS, 100, 4984-4989, 2003

41. Detection of biomolecular binding by using microcantilever • Label-free detection of biomolecules • Methods for measuring the microcantilever bending - Optical Less amenable to monolithic integration and multiplexed detection because of difficulties in laser alignment and power management Interference with turbid or opaque fluidic and smoky environment - Piezoresistive Compatible with aqueous media and parallel cantilever arrays The piezoresistors cover a large length of the cantilever, and high doping levels are required  the stress measurement is not localized  thermal and electronic noise, thermal drift, non-linearity in piezo- response More than 50 nm bending is required

42. Nanomechanical cantilever arrays Linear position detector : beam-deflection With an accuracy of 0.1 nm Mckendry et al., PNAS, 99, 9783-9788 (2002)

43. MOSFET-Embedded Microcantilever • Embedding a metal-oxide semiconductor FET(MOSFET) into the base of the cantilever, and recording decreases in drain current with deflection as small as 5 nm. • The specific biomolecular binding between ligands and receptors on the surface of a microcantilever beam  physical bending of the beam  change in the surface stress  altered channel resistance modulation of the channel current underneath the gate region. • Embedding of MOSFET in the high-stress region of the cantilever to measure deflection. Shekhawart et al., Science, 17, 1592-1595 (2006)

44. N-type FET

45. Molecular imaging Biomedical Sciences : - Ultra-sensitive imaging of biological targets under non-invasive in-vivo conditions - Fluorescence, positron emission tomography, Magnetic resonance imaging - Ultra-sensitive imaging - Cancer detection, cell migration, gene expression, angiogenesis, apotosis - MRI : powerful imaging tool as a result of non-invasive nature, high spatial resolution and tomographic capability Resolution is highly dependent on the molecular imaging agents  signal enhancement by using contrast agents : iron oxide nanoparticles

47. In-vivo MR detection of cancer using NP-Herceptin conjugates Detection of tumors as small as ~ 50 mg cells

48. Virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodesNam et al., Science, 312, 885-888, 2006 • Smaller and more flexible Li ion batteries • Dimensionally small batteries : Nanoparticles, nanotubes, nanowires as well as several assembly methods based on lithography, block copolymer, layer-by-layer deposition • Nanostructured materials : Improvement of the electrochemical property of Li ion batteries • Monodisperse, homogeneous nanomaterials and hierarchical organization control are needed to maximize the potential

49. M13 virus • Helically wrapped by ~ 2700 major coat proteins(p8) and minor coat proteins ( p3, p6, p7, and p9) around its single-stranded DNA • Coat proteins were used to form functional hetero-structured template for precisely positioned nano-materials

50. Predictive-based design Fusion of tetraglutamate(EEEE-) to the N-terminus of the major coat p8 protein - A general template for growing nanowires through the interaction of the glutamate with various metal ions - A blocking motif for gold nanoparticles due to the electrostatic repulsion, reducing nonspecific gold NP binding to phage Favorable interaction with the positively charged electrolyte polymer (Ex) Design of cobalt oxide nanowires - Incubation of the virus templates in aqueous cobalt chloride solution - Cobalt oxide has large reversible storage capacity arising from displacement reactions : ~ three times larger capacity compared to carbon-based anodes currently used in commercial batteries - Homogeneous and high-crystalline nanowires : 141.7 m2/g